PROJECT 3 SUMMARY Understanding the earliest events in Alzheimer disease (AD) has the potential to lead to therapeutic strategies that slow and/or halt disease progression. Addressing AD at its earliest stages is critical as it might be too late to regain cognitive function once neuronal loss has occurred. Development of neurofibrillary tangles (NFT) in the entorhinal cortex (EC) is among the earliest pathological events in AD, but what underlies this regional vul- nerability remains a mystery. NFT are abnormal aggregates of the microtubule-associated protein tau that be- come hyperphosphorylated and aggregated following abnormal kinase activity. Several post-translational modi- fications have been proposed to contribute to tau toxicity, with hyperphosphorylation thought to play a pivotal role. Recently, it has become widely recognized that late-onset AD is associated with abnormalities in mi- croRNAs (miRNAs), small non-coding RNAs that are powerful regulators of gene expression. MiRNAs bind to and silence target mRNAs on the post-transcriptional level. Each miRNA can bind many target transcripts, providing an additional level of regulation that complements canonical transcriptional pathways. While a con- sensus is beginning to emerge that certain miRNAs are consistently altered in AD, the functional significance if these findings remain unclear. The overall objective of this application is to characterize miRNA regulation of the expression of tau and glycogen synthase kinase 3 b (GSK3b), a kinase implicated in tau phosphorylation and leading candidate drug target in AD. This will be accomplished by performing miRNA profiling studies in human autopsy brain tissue and then modeling candidate pathogenic changes with in vitro mammalian neu- ronal cultures and transgenic Drosophila. Our central hypothesis is that down-regulation of miRNAs leads to de-repression of a genetic program that increases expression of genes that drive tau toxicity. To test our hy- pothesis, we propose the following specific aims: 1) to characterize changes in miRNA expression early in AD progression, with a focus on the lateral entorhinal cortex, a region affected early in AD, 2) to characterize miR- NA regulation of the expression of tau and GSK3b, and 3) to demonstrate that miRNAs modulate tau neurotox- icity. This project is significant because it will lead to a better understanding of a genetic program that regulates the expression of genes that we hypothesize contribute the generation of toxic forms of hyperphosphorylated tau and the progression of AD. This proposal is innovative because looking at miRNA regulation has forced a re-assessment of gene expression that will provide a new perspective on the pathogenesis of AD as well as a new framework and new models to investigate mechanisms of tau regulation and toxicity. Together, these ex- periments will help us address the critical need of understanding the earliest events in AD and help pave the way towards new strategies to address AD with miRNA-based biomarkers and therapeutics.